1. FIELD OF THE INVENTION
The present invention relates to a method for biochemical assay and an analyzer used for the method. More particularly, the present invention relates to a method for biochemical assay which comprises converting objective components into appropriate derivatives, separating the derivatives into respective components and assaying the components and to an analyzer used for the method.
2. STATEMENT OF THE PRIOR ART
In analysis of biochemical components by means of liquid chromatography, the objective components are often present in a trace amount and hence, some methods have been hitherto provided for purposes of detecting the trace component in high sensitivity, as will be described below. One is a method involving derivatization in which various reagents for derivatization are reacted with the objective components to convert into more readily detectable derivatives. The derivatization method in liquid chromatography is roughly classified into a pre-column labeling procedure in which derivatization is performed prior to separation with a separation column and a post-column labeling procedure in which derivatization is performed after separating into respective components using a separation column. Choice of either the pre-column labeling or the post-column labeling procedure upon derivatization depends on conditions of the reaction. As described in "ANALYSIS", February, 1987, pages 100-105, for example, in case that a rate of reacting with reagents for derivatization is slow or pressure must be applied, or, in case that gas generates or in case that interference in detection occurs with reagents for derivatization, etc., a modified pre-column labeling method is used; the modified method comprises performing derivatization of a sample in the system independently from an analyzer and performing separation and quantitative determination by liquid chromatography. On the other hand, in case that the reaction is not accompanied by the aforesaid problems, there is applied a pre-column labeling method in which a sample is injected into an analyzer and derivatization is conducted in a reaction coil to achieve separation and quantitative assay, or a post-column labeling method in which a reagent for derivatization is introduced into an analyzer after separation by liquid chromatography. In the post-column labeling method, great restrictions are imposed as compared to the pre-column labeling method because the reaction is caused in an eluate; however, the method has been widely adopted for biochemical analyzers mainly comprised of liquid chromatography such as an amino acid analyzer, a catecholamine analyzer, etc., since the method can be easily automated. Further, in the pre-column labeling method, reaction conditions can be set independently from conditions for analyzers and hence, a derivatization procedure coupled with a pretreatment or concentration is also utilized.
Apart from the trend that demands high sensitivity for detection of a trace component, development has also been extensively made on ultra high performance liquid chromatography using finely divided fillers or on micro liquid chromatography using a micro column. Effects attained by such analyzers are described in detail in J. Chromatogr. Library, "The Science of Chromatography", 32, 435-447 (1985). These ultra high performance liquid chromatography and micro liquid chromatography are considered to be one course of future liquid chromatography. To use a post-column labeling method in the case of assembling a biochemical analyzer by applying ultra high performance liquid chromatography or micro liquid chromatography leads to dilution of the objective components upon derivatization of the objective components and broadening peaks in detection or reduction in detection sensitivity due to noise in pulsation of a pump for feeding a reagent for derivatization, etc. thereby to sacrifice effects to be achieved by ultra high performance liquid chromatography and micro liquid chromatography. Standing on such a viewpoint, biochemical assay and an analyzer used for the assay have been investigated based on liquid chromatography adopting the pre-column labeling procedure to apply to analysis of amines, amino acids, aldehydes, etc. Among these methods, however, there are few methods in which objective components are converted into derivatives in a reaction coil of the analyzer system after a sample is injected into an analyzer, as shown in the "ANALYSIS" publication supra. This is because it takes a long time for derivatization of the objective components in a reaction coil, as compared to a time period required for separation and detection in liquid chromatography and therefore, in the case of treating many samples, it is advantageous to perform a derivatization step by a serial treatment outside the system, not in a reaction coil. Such biochemical assay is shown in J. Chromatogr., 344, 61-70 (1985) or Anal. Biochem., 155, 28-33 (1986). This assay is concerned with analysis of catecholamines with higher sensitivity in a simpler manner than in a conventional method; derivatization of catecholamines is performed outside the analyzer system, since it generally takes a time period as long a 40 minutes. On the other hand, a pre-column labeling method discussed in Anal. Biochem., 133, 330-335 (1983) is also known. In this case, pretreatment of a sample such as removal of protein from the sample, or the like is needed. In addition, it is difficult to uniformly mix the objective components in the sample with a reagent for derivatization.
In Published Examined Japanese Patent Application (KOKOKU) No. 62-56460, there is described liquid chromatography using a pre-column labeling apparatus used for derivatization of objective components which comprises introducing a sample into an adsorption column packed with a filler for adsorbing the objective components in the sample to adsorb the objective components onto the filler and then introducing a labeling agent into the adsorption column to convert the objective components into derivatives thereof. In this pre-column labeling apparatus, however, a rotary column is used as the adsorption column, its assembly is extremely complicated and a long period of time is required for analysis. Further the publication supra fails to disclose analysis of catecholamines.
As mentioned above, the prior art described above does not take into consideration shortening a time period for analysis including derivatization and improvement in accuracy of analysis. Further in the case that a derivatization reaction is serially performed for purposes of increasing the number of samples to be treated, a time period from the reaction to detection varies depending upon sample so that the prior art methods encounter the problem that analytical accuracy is lowered.